Apparatus and method for detecting and identifying microorganisms

ABSTRACT

The present invention is an apparatus for detecting the presence, quantity and identity of one or more microorganisms in a sample and a method for using the same. The apparatus is composed of one or more chambers and a sensing element for sensing microorganisms. In particular embodiments, the sensing element is an array of chemoresponsive dyes deposited on a substrate in a predetermined pattern combination, wherein the combination of the dyes have a distinct and direct spectroscopic, transmission, or reflectance response to distinct analytes produced by the microorganism which is indicative of the presence, quantity and identity of the microorganism.

INTRODUCTION

This application is a continuation of U.S. Ser. No. 14/471,585 filedAug. 28, 2014, which is a continuation of U.S. Ser. No. 11/870,670 filedOct. 11, 2007, now issued as U.S. Pat. No. 8,852,504, which claims thebenefit of U.S. Provisional Application No. 60/829,025 filed Oct. 11,2006, each of which are herein incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Conventional diagnosis of microbial infections generally relies on cellculturing to detect and identify the microorganism responsible for theinfection. While cell culturing is inexpensive, it can be relativelyslow because it relies on visual detection of individual bacterialcolonies (i.e., ˜10⁶ bacteria). For example, while colonies of a fastgrowing bacterium can be observed between ˜24 to 48 hours, slowergrowing bacteria require incubation periods of a week or more to detectbacterial colonies. As further alternatives, instruments have beendeveloped using various principles of detection including infrared orfluorescence spectroscopy, bioluminescence, and flow cytometry (Basile,et al. (1998) Trends Anal. Chem. 17:95-109; Bird, et al. (1989) RapidSalmonella Detection by a Combination of Conductance and ImmunologicalTechniques; Blackwell Sci. Publications: Oxford, Vol. 25; Fenselau, Ed.(1994) Mass Spectrometry for the Characterization of MicroorganismsWashington D.C., Vol. 240; Lloyd, Ed. (1993) Flow Cytometry inMicrobiology; Springer-Verlag London Limited: Germany; Perez, et al.(1998) Anal. Chem. 70:2380-2386; Wyatt (1995) Food Agri. Immunol.7:55-65). Among these, the primary physical/chemical methods ofbacterial detection are those which involve the detection of somenaturally occurring component of the bacterium.

BACT/ALERT uses, for example, a colorimetric sensor detection systemwhich detects microorganism growth by the production of CO₂. When theCO₂ levels reach a certain level, the sensor turns yellow giving apositive result for bacteria present. This system can be used for a widevariety of microorganisms and has a success rate of 95% in 24 hours and98% in 72 hours (Weinstein, et al. (1995) J. Clin. Microbiol.33:978-981; Wilson, et al. (1995) J. Microbiol. 33:2265-2270; Wilson, etal. (1992) J. Clin. Microbiol. 30:323-329).

Other devices and methods for detecting microorganisms are provided inU.S. Pat. Nos. 5,094,955; 6,777,226; 6,197,577; 5,976,827; and5,912,115. In general, these devices rely on the use of a single sensor(e.g., pH or carbon dioxide indicator) in a layer adjacent to a layer ofgrowth medium for detecting the presence of a bacterium.

Bacterial identification methods usually include a morphologicalevaluation of microorganisms as well as tests for the organism's abilityto grow in various media sources under various conditions. Thesetechniques allow for the detection of single organisms, however,amplification of the signal is required through growth of a single cellinto a colony and no single test provides a definitive identification ofan unknown bacterium. Traditional methods for the identification ofbacteria involve pre-enrichment, selective enrichment, biochemicalscreening, and serological confirmation (Tietjen & Fung (1995) Crit.Rev. Microb. 21:53-83; Kaspar & Tartera (1990) Methods Microbiol.22:497-530; Helrich (1990) Official Methods of Analysis of Associationof Official Analytical Chemists; 15 ed.; AOAC: Arlington, Va., Vol. 2;Hobson, et al. (1996) Biosensors & Bioelectronics 11:455-477).

Alternative methods such as immunoassays and PCR-based approaches havebeen pursued with varying degrees of success (Iqbal, et al. (2000)Biosensors & Bioelectronics 15:549-578; Morse (2000) DetectingBiological Warfare Agents; Lynne Rienner Publishers, Inc: Boulder,Colo.). However, in the case of PCR, such an approach is expensive andrequires pure samples, hours of processing, and an expertise inmicrobiology (Spreveslage, et al. (1996) J. Microbiol. Methods26:219-224; Meng, et al. (1996) Intl. J. Food Microbiol. 32:103-113). Analternative method, gas chromatography/mass spectrometry (GC/MS), hasbeen used to produce a fatty acid profile or “fingerprint” for thedetection and identification of microorganisms (Swaminathan & Feng(1994) Ann. Rev. Microbiol. 48:401-426).

Array-based vapor sensing is an approach toward the detection ofchemically diverse analytes. Based on cross-responsive sensor elements,rather than specific receptors for specific analytes, these systemsproduce composite responses unique to an odorant in a fashion similar tothe mammalian olfactory system (Stetter & Pensrose, Eds. (2001)Artificial Chemical Sensing: Olfaction and the Electronic Nose;Electrochem. Soc.: New Jersey; Gardner & Bartlett (1999) ElectronicNoses: Principles and Applications; Oxford University Press: New York;Persuad & Dodd (1982) Nature 299:352; Albert, et al. (2000) Chem. Rev.100:2595-2626; Lewis (2004) Acc. Chem. Res. 37:663-672; James, et al.(2005) Microchim. Acta 149:1-17; Walt (2005) Anal. Chem. 77:45A). Insuch arrays, one receptor responds to many analytes and many receptorsrespond to any given analyte. A distinct pattern of responses producedby the colorimetric sensor array provides a characteristic fingerprintfor each analyte. Using such systems, volatile organic compounds havebeen detected and differentiated (Rakow & Suslick (2000) Nature406:710-713; Suslick & Rakow (2001) Artificial Chemical Sensing:Olfaction and the Electronic Nose; Stetter & Penrose, Eds.; Electrochem.Soc.: Pennington, N.J.: pp. 8-14; Suslick, et al. (2004) Tetrahedron60:11133-11138; Suslick (2004) MRS Bulletin 29:720-725; Rakow, et al.(2005) Angew. Chem. Int. Ed. 44:4528-4532; Zhang & Suslick (2005) J. Am.Chem. Soc. 127:11548-11549).

Array technologies of the prior art generally rely on multiple,cross-reactive sensors based primarily on changes in properties (e.g.,mass, volume, conductivity) of some set of polymers or onelectrochemical oxidations at a set of heated metal oxides. Specificexamples include conductive polymers and polymer composites (Gallazzi,et al. (2003) Sens. Actuators B 88:178-189; Guadarrana, et al. (2002)Anal. Chim. Acta 455:41-47; Garcia-Guzman, et al. (2003) Sens. ActuatorsB 95:232-243; Burl, et al. (2001) Sens. Actuators B 72:149-159; Wang, etal. (2003) Chem. Mater. 15:375-377; Hopkins & Lewis (2001) Anal. Chem.73:884-892; Feller & Grohens (2004) Sens. Actuators B 97:231-242;Ferreira, et al. (2003) Anal. Chem. 75:953-955; Riul, et al. (2004)Sens. Actuators B 98:77-82; Sotzing, et al. (2000) Anal. Chem.72:3181-3190; Segal, et al. (2005) Sens. Actuators B 104:140-150; Burl,et al. (2002) Sens. Actuators B 87:130-149; Severin, et al. (2000) Anal.Chem. 72:658-668; Freund & Lewis (1995) Proc. Natl. Acad. Sci. U.S.A.92:2652-2656; Gardner, et al. (1995) Sens. Actuators B 26:135-139;Bartlett, et al. (1989) Sens. Actuators B 19:125-140; Shurmer, et al.(1990) Sens. Actuators B 1:256-260; Lonergan, et al. (1996) Chem. Mater.8:2298-2312), polymers impregnated with a solvatochromic dye orfluorophore (Chen & Chang (2004) Anal. Chem. 76:3727-3734; Hsieh &Zellers (2004) Anal. Chem. 76:1885-1895; Li, et al. (2003) Sens.Actuators B 92:73-80; Albert & Walt (2003) Anal. Chem. 75:4161-4167;Epstein, et al. (2002) Anal. Chem. 74:1836-1840; Albert, et al. (2001)Anal. Chem. 73:2501-2508; Stitzel, et al. (2001) Anal. Chem.73:5266-5271; Albert & Walt (2000) Anal. Chem. 72:1947-1955; Dickinson,et al. (1996) Nature 382:697-700; Dickinson, et al. 1996) Anal. Chem.68:2192-2198; Dickinson, et al. (1999) Anal. Chem. 71:2192-2198), mixedmetal oxide sensors (Gardner & Bartlett (1992) Sensors and SensorySystems for an Electronic Nose; Kluwer Academic Publishers: Dordrecht;Zampolli, et al. (2004) Sens. Actuators B 101:39-46; Tomchenko, et al.(2003) Sens. Actuators B 93:126-134; Nicolas & Romain (2004) Sens.Actuators B 99:384-392; Marquis & Vetelino (2001) Sens. Actuators B77:100-110; Ehrmann, et al. (2000) Sens. Actuators B 65:247-249; Getino,et al. (1999) Sens. Actuators B 59:249-254; Heilig, et al. (1997) Sens.Actuators B 43:45-51; Gardner, et al. (1991) Sens. Actuators B4:117-121; Gardner, et al. (1992) Sens. Actuators B 6:71-75; Corcoran,et al. (1993) Sens. Actuator B 15:32-37; Gardner, et al. (1995) Sens.Actuators B 26:135-139), and polymer coated surface acoustic wave (SAW)devices (Grate (2000) Chem. Rev. 100:2627-2648; Hsieh & Zellers (2004)Anal. Chem. 76:1885-1895; Grate, et al. (2003) Anal. Chim. Acta490:169-184; Penza & Cassano (2003) Sens. Actuators B 89:269-284; Levit,et al. (2002) Sens. Actutors B 82:241-249; Grate, et al. (2001) Anal.Chem. 73:5247-5259; Hierlemann, et al. (2001) Anal. Chem. 73:3458-3466;Grate, et al. (2000) Anal. Chem. 72:2861-2868; Ballantine, et al. (1986)Anal. Chem. 58:3058-3066; Rose-Pehrsson, et al. (1988) Anal. Chem.60:2801-2811; Patrash & Zellers (1993) Anal. Chem. 65:2055-2066).However, the sensors disclosed in these prior art references do notprovide a diversity of interactions with analytes; interactions arelimited to the weakest and least specific of intermolecularinteractions, primarily van der Waals and physical adsorptioninteractions between sensor and analyte. As such, both sensitivity fordetection of compounds at low concentrations relative to their vaporpressures and selectivity for discrimination between compounds iscompromised with these prior art sensors.

Cross-responsive sensor technologies have also been applied to theidentification of bacteria (Lai, et al. (2002) Laryngoscope 112:975-979;McEntegart, et al. (2000) Sensors and Actuators B 70:170-176; Gibson, etal. (1997) Sensors and Actuators B 44:413-422; Ivnitski, et al. (1999)Biosensors & Bioelectronics 14:599-624). These cross-responsive sensortechnologies have employed a variety of chemical interaction strategies,including the use of conductive polymers (Freund & Lewis (1995) Proc.Natl. Acad. Sci. USA FIELD Publication Date 92:2652-2656), conductivepolymer/carbon black composites (Lonergan, et al. (1996) Chem. Mater.8:2298-2312), fluorescent dye/polymer systems (Walt (1998) Acc. Chem.Res. 31:267-278), tin oxide sensors (Heilig, et al. (1997) Sensors andActuators, B: Chemical B43:45-51), and polymer-coated surface acousticwave (SAW) devices (Grate (2000) Chem. Rev. 100:2627-2647). For example,an array of four metal oxide sensors has been used to detect andidentify six pathogenic bacteria by sampling the headspace over thegrowing microorganisms, wherein the sensor correctlyidentified/classified 62% of the pathogens (Craven, et al. (1994) NeuralNetworks and Expert Systems in Medicine and Healthcare; University ofPlymouth: Plymouth). The use of such technologies for medicalapplication has been described (Thaler, et al. (2001) Am. J. Rhinology15:291-295); however, these systems employ the detection of chemicallynon-coordinating organic vapors without exploring the detection of themost toxic and odiferous compounds (e.g., phosphines and thiols). Ingeneral, most cross-responsive sensor devices of the prior art havelimited detection sensitivity and remain quite non-selective (O'Hara(2005) Clin. Microbiol. Rev. 18:147-162).

Additional devices for detecting microorganisms are disclosed in U.S.Pat. Nos. 6,030,828; 4,297,173; 4,264,728; 5,795,773; 5,856,175;6,855,514; 7,183,073; and U.S. Patent Application No. 2005/0170497.

Needed is a cost-efficient, non-invasive, sensitive and selective sensorwhich can detect, quantify and discriminate between microorganisms. Thepresent invention meets this long-felt need.

SUMMARY OF THE INVENTION

The present invention is an apparatus for detecting and identifyingmicroorganisms. In one embodiment, the apparatus is composed of a mediumfor supporting growth of a microorganism and at least one colorimetricsensing element placed in or proximate to the medium, wherein saidsensing element is composed of an array which has a plurality ofchemoresponsive dyes deposited thereon in a predetermined patterncombination, wherein the combination of the dyes have a distinct anddirect spectroscopic, transmission, or reflectance response to distinctanalytes produced by the microorganism which is indicative of thepresence and identity of the microorganism. In accordance with thisembodiment, the apparatus further contains an air flow means, or is partof a system which includes a visual imaging means, with some embodimentsincluding an aerating means.

In another embodiment, the apparatus of the invention is composed of afirst chamber for culturing a cell, and a second chamber with a sensingelement disposed therein, wherein the first chamber and second chamberare separated by a gas impermeable barrier produced from a materialselected for being permeabilized. In accordance with this embodiment,the apparatus can further include a medium for supporting growth of amicroorganism.

Kits and methods for detecting, quantifying or identifying amicroorganism using the apparatus of the invention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are illustrations of an apparatus of the invention. FIG. 1Ais an exploded side view of the apparatus. FIG. 1B shows a side view ofthe assembled apparatus. FIG. 1C shows a perspective view of theassembled apparatus with the colorimetric sensing element placed printside down in the bottom of a Petri dish container (i.e., surface ofarray with chemoresponsive dye is visible from the outside).

FIGS. 2A-2H show illustrations of an apparatus of the invention in theform of a liquid/blood culture bottle. In the embodiment depicted inFIG. 2A, the medium is in the bottom of the bottle and the colorimetricsensing element is in the headspace above the medium. In the embodimentdepicted in FIG. 2B, the colorimetric sensing element is placed in thebottom of the bottle and the medium is separated from the colorimetricsensing element by a gas-permeable membrane. In the embodiment depictedin FIG. 2C, the colorimetric sensing element is composed of dyessuspended in a gas-permeable matrix at the bottom of the bottle and themedium is in the main body of the bottle. FIG. 2D depicts embodimentswherein the culture medium is separated from the growth medium by a gasimpermeable barrier produced from a material selected for being capableof being permeabilized. FIGS. 2E and 2F depict the apparatus of FIG. 2D,wherein the gas impermeable barrier has been ruptured (FIG. 2E) or madegas permeable (FIG. 2F). FIG. 2G illustrates an overlapping radial sealwhile FIG. 2H illustrates a capping seal at the bottom of the container.

FIG. 3 depicts a colorimetric sensing element in a petri dish containerconfigured for recirculating flow of headspace. The arrows indicatedirection of gas flow.

FIGS. 4A-4B shows an apparatus of the invention employing a liquid orsemi-liquid medium, wherein the colorimetric sensing element ispositioned proximate to the medium (FIG. 4A) as compared to being placedin the medium (FIG. 4B).

FIG. 5 depicts intermolecular interactions on a semi-quantitative energyscale.

FIGS. 6A-6B show an integrated system for detecting and identifyingmicroorganisms, wherein the apparatus is placed in a growth chamberequipped with an imaging system. Image and data analysis are performedby a computer. The integrated system can be stationary (FIG. 6A) orprovide an aerating means for moving the apparatus thereby aerating andhomogenizing the microorganisms in culture (FIG. 6B).

FIG. 7 shows the classification of several species of bacteria(Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli)using hierarchical cluster analysis.

FIG. 8 shows results of linear discriminant analysis for multiplestrains of E. coli.

FIG. 9 shows the response of select color channels during the growth of1020 cfu of E. coli.

FIG. 10 shows the detection times for representative loadings of E.coli.

FIG. 11 shows growth curves obtained with colorimetric sensing elementsfor E. coli, P. aeruginosa, S. pyogenes, S. aureus, and M. catarrhalison solid tryptic soy agar with 5% sheep blood.

FIG. 12 shows a sample of data collected from blood culture experiments,wherein each line represents a different color channel.

FIG. 13 shows the second derivative of color channel B28, as a functionof time, from colorimetric sensing element signal arising from lowloadings of E. coli (i.e., initial cell concentrations of 500 cfu/mL to4000 cfu/mL).

FIG. 14 depicts colorimetric sensing element response patterns for E.coli, S. aureus, E. faecalis, and P. aeruginosa grown in blood culture.

DETAILED DESCRIPTION OF THE INVENTION

An apparatus and method for using the same are provided which can beadvantageously used to detect and identify microorganisms in variousapplications, including but not limited to disease diagnosis, qualitycontrol, and environmental contamination. In particular embodiments, theinvention relates the use of chemoresponsive dyes, such as Lewisacid/base dyes (e.g., metalloporphyrin), Brønsted acidic or basic dyes(e.g., pH indicators) and dyes with large permanent dipoles (e.g.,zwitterionic solvatochromic dyes), for detecting and identifyingmicroorganisms based upon analytes produced by the microorganisms.

As depicted in FIG. 1, the instant apparatus 10 is a container in theform of, e.g., a petri dish, with at least one interior side 20 (e.g.,the top side) containing a medium 30 for supporting growth of at leastone microorganism in a sample, and the opposite interior side 22 (e.g.,the bottom side) having affixed thereto a sensing element 40, which whenin use is placed in or proximate to medium 30. In particularembodiments, the sensing element is not in contact with the surface ofthe medium.

Additional features of the instant apparatus can include a support means50 and retaining means 60 (FIG. 1) for positioning sensing element 40 inthe headspace of microorganisms growing on or in medium 30. It iscontemplated that the bottom 22 of the interior side of apparatus 10 canbe modified to provide support means 50 for holding sensing element 40.Support means 50 can be integral with apparatus 10, wherein in usesensing element 40 is placed on support means 50 and retainer means 60holds sensing element 40 in place on support means 50 and prevents itfrom moving. Retainer means 60 can snap in place, be chemically orultrasonically welded in place, glued in place, etc. While someembodiments embrace a removable sensing element 40, other embodimentsprovide that sensing element 40 is permanently fixed to support means50, thereby obviating the need of retainer means 60.

For the purpose of disclosing the instant apparatus, the terms “top” and“bottom” are used herein to describe two opposing sides and are not tobe construed as limiting the instant apparatus to any particularorientation or configuration. In this regard, other configurations suchas bottles or vials are also embraced by the present invention. By wayof illustration, the configuration depicted in FIG. 2A shows the instantapparatus in the form of a bottle, wherein medium 30 (e.g., a bloodculture medium) is in the bottom 22 of apparatus 10 with sensing element40 positioned at the top 20 of apparatus 10 via support means 50attached to a cap or lid 55. In the bottle configuration depicted inFIG. 2B, medium 30 is in a first chamber 70 of apparatus 10 with sensingelement 40 located in a second chamber 72 (and optionally affixed to thebottom 22 of apparatus 10), wherein the first chamber 70 and secondchamber 72 are separated by a gas-permeable barrier 80 a. Alternatively,the embodiment shown in FIG. 2C provides for the direct application ofsensing element 40 to the bottom 22 of apparatus 10 in the form ofsensing dyes suspended in gas-permeable matrix 42. Thus, while sensingelement 40 depicted in FIG. 2A can be monitored from the side ofapparatus 10, sensing element 40 depicted in FIGS. 2B and 2C can bemonitored from the bottom 22 of apparatus 10.

In yet another embodiment of the invention, a dual-chamber apparatus 10is provided, wherein the chambers are separated by a gas impermeablebarrier. As illustrated in FIG. 2D, the first chamber 70 of apparatus 10holds media 30, whereas the second chamber 72 contains the sensingelement 40 interfaced to a transparent area such that the sensingelement 40 can be viewed from outside apparatus 10. First chamber 70 andsecond chamber 72 are separated by gas impermeable barrier 80 b so thatsensing element 40 is not exposed to media 30 or other components withinthe first chamber 70 until a user-designated time point. At that point,the baseline value of the sensing element 40 can be measured (e.g.,color, pressure, fluorescence or other signal), barrier 80 b can beruptured or otherwise made permeable, and changes in sensor element 40characteristics can be monitored, e.g., at regular time pointsthereafter. In accordance with this configuration of the instantapparatus, gas impermeable barrier 80 b can be permeabilized byrupturing or breaking barrier 80 b (e.g., via mechanical puncturingusing a device equivalent to a needle or nail that is inserted throughthe top 20 of apparatus 10) to expose sensing element 40 to the media 30or headspace above the media. In this regard, FIG. 2E depicts theinversion of apparatus 10 so that sensing element 40 is in the headspaceabove medium 30 after permeabilization of gas impermeable barrier 80 b.Alternatively, gas impermeable barrier 80 b can be permeabilized togases (and therefore structurally intact) at a user-designated timepoint and the container maintained in an upright posture (see FIG. 2F).For example, it is contemplated that gas permeability of a gasimpermeable barrier 80 b can be modified by exposure to ultrasound;applied electric field; a change in temperature, pH, humidity, etc. Byway of illustration, Ito, et al. ((1997) Die Angewadndte MakromolekulareChemie 248:85-94), teach that chitosan membrane has a low permeabilityfor gases in its dry state; however, permeability to gases such ascarbon dioxide increases upon exposure to water vapor. Similarly,Kulshrestha, et al. ((2005) Bull. Mater. Sci. 28:643-646) teach that thepermeability of polyethersulphone membranes to H₂ and CO₂ increased withincreasing temperature.

As can be appreciated by the skilled artisan, the configuration shown inFIG. 1 may be more suitable for solid or certain semi-solid mediawhereas the configurations shown in FIG. 2 may be more suitable forliquid or semi-liquid media.

In some embodiments, all or a portion of the container is transparent ortranslucent such that changes in the sensing element 40 can be detected.In particular embodiments, the portion of apparatus 10 containingsensing element 40 is preferably transparent for viewing/imaging changesin sensing element 40 due to microorganism growth and production ofanalytes.

Though the apparatus can be open such that the interior air is exchangedwith the exterior air, it is desirable that the apparatus be a sealed orsealable container. The container may be sealed by snapping or screwingthe top and bottom sides together or by using a permeable orsemi-permeable sealing film (e.g., PARAFILM) or any combination thereof(or any other sealing device). Alternatively, a plug or stopper can beemployed, e.g., in configurations employing bottle-shaped containers.The container itself can be composed of either a gas-permeable or agas-impermeable material, depending on the growth requirements of themicroorganism. Different container composition materials can includelaminates (gas impermeable and/or hydrophobic gas-permeable membranes)or other suitable materials well-known in the art (e.g., glass) forproducing culture plates, vials or bottles. Moreover, the container canbe modified with vents (e.g., a gas permeable membrane in an opening ofthe container wall).

In accordance with embodiments pertaining to a dual-chambered apparatus,it is contemplated that apparatus 10 can be produced by an overlappingradial seal (see FIG. 2G) or by employing a capping seal at the bottomof the apparatus 10 (FIG. 2H) to generate the second chamber 72 housingsensing element 40.

As illustrated in FIG. 1, FIG. 2A and FIG. 2E, sensing element 40 ispositioned in the headspace above medium 30 in such a manner that thevolatile analytes generated by the microorganisms reach sensing element40 by simple diffusion. However, an alternative embodiment shown in FIG.3 provides an integrated, recirculating gas (air) pump 70 to establish adynamic flow of headspace across sensing element 40 such that theresponse time of the sensing element 40 to the analytes can be reduced.

Furthermore, some embodiments embrace sensing element 40 positioned inthe headspace (see, e.g., FIGS. 2A, 2E and 4A), while other embodimentsembrace a colorimetric sensing element in contact with or placed in themedium. By way of illustration, FIG. 4B shows medium 30 in the bottom 22of apparatus 10 with colorimetric sensing element 40 affixed via supportmeans 50 to the top 20 of apparatus 10 and positioned in medium 30 whenin use.

As the skilled artisan can appreciate, a variety of different media canbe employed in the instant invention. The growth medium of the instantapparatus can be a solid, semi-solid, or liquid formulation thatcontains nutrients for supporting growth of one or more microorganisms.As used herein, a solid medium is defined as any formulation that holdsits own form or shape, at least for a few minutes, and hence includesgelatin growth medium. In one embodiment, the growth medium is preformedand the sample subsequently applied to the medium. For example, a liquidsample could be applied onto an already gelled medium or onto adehydrated or partially dehydrated gel medium so as to immobilize themicroorganisms on the surface of the medium. Alternatively, the sampleitself can be a liquid, semi-solid or semi-liquid (e.g., paste or gel)and used in the preparation of the medium or a layer of the medium. Forexample, a liquid sample can be mixed with a dry powdered gelling agentto form a solid or semi-solid matrix which is applied to the surface ofa preformed growth medium before gelling has occurred. As a furtheralternative, the sample is applied to the surface of the growth mediumon a non-gel absorbent material, such as a sponge material, cellulose,glass fiber, filter paper, etc.

Semi-solid or solid media can be prepared using any suitable gellingagent or combination of gelling agents including natural and synthetichydrogel powders, gums, agars, agaroses, carageenans, bentonite,alginates, collagens, gelatins, fused silicates, water soluble starches,polyacrylates, celluloses, cellulose derivatives, polyethylene glycols,polyethylene oxides, polyvinyl alcohols, dextrans, polyacrylamides,polysaccharides or any other gelling or viscosity enhancing agents.

The nutritional components that make up a complex microbial mediuminfluence the metabolic pathways used by microorganisms. Organic acids,bases and various analytes are produced in proportions dependent on thenutrients available. These metabolic products also vary from species tospecies of microorganism and can advantageously be detected and used inthe identification of one or more microorganisms in a sample. The amountof metabolites produced by microorganisms increases with increasedgrowth time and increased initial concentration. The amount ofmicroorganism present depends on the type of microorganism, its growthrate, the medium, and the growth environment. The type and amount ofmetabolites produced by microorganisms depend on all of the abovefactors as well as the growth phase of the microorganism. Accordingly,the medium of the instant invention is added to provide nutrients forthe growth of microorganisms so that colorimetric sensingelement-detectable analytes are produced. Many types of media arewell-known in the art for different types of microorganisms. Forexample, for supporting the growth of an aerobic organism, the media caninclude, e.g., tryptone, soytone, proteose peptone, malt extract,dextrose and MOPS. If the microorganism is an anaerobic organism, themedia can further include the media listed above for aerobic organisms,as well as Hemin, L-cystine and Menadione. For coliforms, the media caninclude, e.g., Lactose, bile salts #3, K₂HPO₄, KH₂PO₄, (NH₄)₂SO₄),MgSO₄, Na-bisulfide and SDS. For yeast, mold and other acid tolerantmicroorganisms, the media can include, e.g., dextrose, yeast extract,(NH₄) citrate and tartaric acid to a pH of 5.5. Liquid culture media,including blood culture media, is also encompassed within the scope ofthe invention.

The medium can also contain conditioning components, such as lyticagents, lytic enzymes, antibiotic neutralizers, surfactants or othermaterials helpful for improving microorganism detection capabilities.Alternatively, conditioning components can be combined with the sampleor in a separate layer of medium.

Lytic agents for conditioning can be added for lysing blood cells in thesample, for allowing for a smoother gel, and/or for better rehydrationof the gel. Examples of possible lytic agents include saponin,digitonin, TWEEN, polysorbitan monolaurate, and other surfactants. Lyticenzymes, typically though not necessarily proteolytic enzymes, can beadded for digesting cellular material in a blood sample, for making asmoother gel, and/or for better rehydration of the gel. The lyticenzymes for conditioning can include one or more proteases, for examplean enzyme mixture derived from Aspergillus oryzae, or the like.

Antibiotic neutralizers can be added for conditioning, in particular forfaster and/or better recovery of microorganisms in the sample. One ormore of such neutralizers could be selected from resins, gums, andcarbon-based materials (e.g., activated charcoal or ECOSORB), or one ofa variety of enzymes to specifically degrade various antibiotics (e.g.,beta lactamase).

A variety of different sensor types can also be used in this invention.While some embodiments embrace the use of conventional pressure, pH, ortemperature sensing elements, particular embodiments of the presentinvention embrace the detection of at least one analyte via a sensingelement composed of an array of chemoresponsive dye spots that changecolor upon contact with various gases or vapors, i.e., a colorimetricsensing element. In this regard, the sensing element can be preselectedto sense one analyte or alternatively sense a plurality of analytes.

A colorimetric sensing element of the present invention is a substratewith a plurality of chemoresponsive dyes deposited thereon in apredetermined pattern combination. The substrate for retaining thechemoresponsive dyes can be the apparatus itself or be composed of anysuitable material or materials, including but not limited to,chromatography plates, paper, filter papers, porous membranes, orproperly machined polymers, glasses, or metals. However, particularembodiments embrace the use of a hydrophobic substrate. Dyes can becovalently or non-covalently affixed in or on a colorimetric sensingelement substrate by direct deposition, including, but not limited to,airbrushing, ink-jet printing, screen printing, stamping, micropipettespotting, or nanoliter dispensing. In embodiments drawn to the apparatusitself for use a substrate, the chemoresponsive dye can be dispersed ina liquid polymer solution, similar to silicon caulking prior to curing.Individual polymer-dye solutions are then placed onto the surface of theapparatus to form an array of chemoresponsive dyes on the surface of theapparatus. See, for example FIG. 2C.

When the sensing element is provided on a substrate which is not theapparatus itself, the sensing element is preferably affixed (e.g., by anadhesive or retaining means) inside a transparent portion of theapparatus so that it is visible from the outside of the apparatus. It iscontemplated that the sensing element can also be placed outside theapparatus, as long as a method is provided that allows the metabolicchanges due to the microorganisms to affect the sensor.

In the illustrative embodiments disclosed in FIGS. 1, 2 and 4, sensingelement 40 is positioned in apparatus with the printed dye surfacefacing to the outside. Sensing element 40 is exposed to analytes inmedium 30 (FIG. 4B) or to analytes present in the headspace above medium30 via support means 50, which can be vented to allow analytes producedby the microorganisms to flow over sensing element 40 (see FIGS. 1, 2A,2E and 4A). As such, sensing element 40 may or may not be flush againstthe inside surface of the apparatus. In embodiments wherein medium 30 isin the background of sensing element 40 when sensing element 40 is beingviewed from the outside of container 20, it is desirable that thesensing element substrate is opaque. By “opaque”, it is meant that thesubstrate sufficiently blocks the viewing or detecting (in any relevantelectromagnetic region) of the sample and/or actual microorganismcolonies in or on the medium from the opposite side of the sensingelement (e.g., semi-opaque, substantially opaque, or fully opaque).Though it is possible to have a transparent or relatively transparentcolorimetric sensing element substrate if the sample is alsosubstantially transparent, it is generally desired that the colorimetricsensing element substrate not be transparent.

In general, the detection and identification of compounds using acolorimetric sensing element is fundamentally based upon supramolecularchemistry and intrinsically relies on the interactions betweenmolecules, atoms, and ions. The classification of inter-molecularinteractions is well-established (FIG. 5) and involves bond formationand coordination, acid-base interactions, hydrogen-bonding,charge-transfer and pi-pi molecular complexation, dipolar and multipolarinteractions, as well as weak interactions such as van der Waalsinteraction and physical adsorption. In contrast to the prior art, theinstant invention advantageously employs chemoresponsive dyes havestrong interactions, e.g., greater than 10 kJ/mol or preferably greaterthan 25 kJ/mol, with analytes produced by microorganisms.

To achieve such strong interactions and further provide a means fordetection, the chemically responsive or chemoresponsive dyes employed inthe instant colorimetric sensing element each contain a center tointeract strongly with analytes, and each interaction center is stronglycoupled to an intense chromophore. As used herein, chemoresponsive dyesare dyes that change color, in either reflected or absorbed light, uponchanges in their chemical environment.

Chemoresponsive dye classes which provide the desired interactions andchromophores include Lewis acid/base dyes (i.e., metal ion containingdyes), Brønsted acidic or basic dyes (i.e., pH indicators), and dyeswith large permanent dipoles (i.e., zwitterionic solvatochromic dyes).The importance of strong sensor-analyte interactions is highlighted byindications that the mammalian olfactory receptors are, in many cases,metalloproteins and that odorant ligation to the metal center isintrinsic to the mechanism of action (Wang, et al. (2003) Proc. Natl.Acad. Sci. U.S.A. 100:3035-3039).

To detect and distinguish a multitude of analytes, the instant apparatuscan employ a plurality of chemoresponsive dyes. In accordance with thepresent invention, the plurality of dyes is deposited on the arraysubstrate in a predetermined pattern combination. Alternatively stated,the dyes are arranged in a two-dimensional spatially-resolvedconfiguration so that upon interaction with one or more analytes, adistinct color and intensity response of each dye creates a signatureindicative of the one or more analytes. A plurality of chemoresponsivedyes encompasses 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or50 individual dyes. In particular embodiments, a plurality ofchemoresponsive dyes is 2 or more, 5 or more, 10 or more, 15 or more, 20or more, 25 or more, or 30 or more dyes. The chemoresponsive dyes can bedeposited in predetermined pattern combinations of rows, columns,spirals, etc., and one or more chemoresponsive dye arrays can be used ina container.

For recognition of analytes with Lewis acid/base capabilities, the useof porphyrins and their metal complexes is desirable. Metalloporphyrinsare ideal for the detection of metal-ligating vapors because of theiropen coordination sites for axial ligation, their large spectroscopicshifts upon ligand binding, their intense coloration, and their abilityto provide ligand differentiation based on metal-selective coordination.Furthermore, metalloporphyrins are cross-responsive dyes, showingresponses to a large variety of different analytes to different degreesand by different color changes.

A Lewis acid/base dye is defined as a dye which has been identified forits ability to interact with analytes by acceptor-donor sharing of apair of electrons from the analyte. This results in a change in colorand/or intensity of color that indicates the presence of the analyte.

Lewis acid/base dyes include metal ion-containing or three-coordinateboron-containing dyes. Exemplary Lewis acids include, but are notlimited to, metal ion-containing porphyrins (i.e., metalloporphyrins),salen complexes, chlorins, bispocket porphyrins, and phthalocyanines.Particularly suitable metal ions complexed with dyes for detectingammonia include Zn(II) and Co(III) metals. In particular embodiments ofthe present invention, the Lewis acid dye is a metalloporphyrin. Forexample, diversity within the metalloporphyrins can be obtained byvariation of the parent porphyrin, the porphyrin metal center, or theperipheral porphyrin substituents. The parent porphyrin is also referredto as a free base porphyrin, which has two central nitrogen atomsprotonated (i.e., hydrogen cations bonded to two of the central pyrrolenitrogen atoms). A particularly suitable parent porphyrin is5,10,15,20-tetraphenylporphyrinate(−2) (TPP dianion), its metalatedcomplexes, its so-called free base form (H₂TPP) and its acid forms(H₃TPP⁺ and H₄TPP⁺²). Suitable metal ion-containing metalloporphyrindyes for use in the apparatus and method of the present inventioninclude, but are not limited to,

2,3,7,8,12,13,17,18-octafluoro-5,10,15,20-tetrakis-(pentafluorophenyl)porphyrinatocobalt(II)[Co(F₂₈TPP)];

2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetraphenylporphyrinatozinc(II)[Zn(Br₈TPP)];

5,10,15,20-tetraphenylporphyrinatozinc(II) [ZnTPP];

5(phenyl)-10,15,20-trikis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrinatozinc(II)[Zn(Si₆PP)];

5,10,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrinatozinc(II)[Zn(Si₈PP];

5,10,15,20-Tetraphenyl-porphyrinatocobalt (II) [CoTPP];

5,10,15,20-Tetrakis(2,6-difluorophenyl)porphyrinatozinc(II) [Zn—F2PP];and

5,10,15,20-Tetrakis(2,4,6-trimethylphenyl)porphyrinatozinc(II) [ZnTMP].

The synthesis of such porphyrins is well-established in the art and isdescribed in U.S. patent application Ser. No. 10/279,788.

A Brønsted acid dye of the present invention is a pH indicator dye whichchanges color in response to changes in the proton (Brønsted) acidity orbasicity of the environment. For example, Brønsted acid dyes are, ingeneral, non-metalated dyes that are proton donors which can changecolor by donating a proton to a Brønsted base (i.e., a proton acceptor).Brønsted acid dyes include, but are not limited to, protonated, butnon-metalated, porphyrins, chlorins, bispocket porphyrins,phthalocyanines, and related polypyrrolic dyes. Polypyrrolic dyes, whenprotonated, are in general pH-sensitive dyes (i.e., pH indicator oracid-base indicator dyes that change color upon exposure to acids orbases)

In one embodiment, a Brønsted acid dye is a non-metalated porphyrin suchas5,10,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrindication [H₄Si₈PP]⁺²; 5,10,15,20-Tetraphenyl-21H,23H-porphine [H₂TPP];or 5,10,15,20-Tetraphenylporphine dication [H₄TPP]⁺². In anotherembodiment of the instant invention, a selected Brønsted dye is anindicator dye including, but not limited to, Bromocresol Purple, CresolRed, Congo Red, Thymol Blue, Bromocresol Green, Nile Red, BromothymolBlue, Methyl Red, Nitrazine Yellow, Phenol Red, Bromophenol Red,Disperse Orange 25, and Bromophenol Blue. As will be appreciated by theskilled artisan, the Brønsted acids disclosed herein may also beconsidered Brønsted bases under particular pH conditions. Likewise, anon-metalated, non-protonated, free base form of a bispocket porphyrinmay also be considered a Brønsted base. However, these dye forms arealso expressly considered to be within the scope of the dyes disclosedherein.

Solvatochromic dyes change color in response to changes in the generalpolarity of their environment, primarily through strong dipole-dipoleand dispersion interactions. To some extent, all dyes inherently aresolvatochromic, with some being more responsive than others. Particularexamples of suitable solvatochromic dyes include, but are not limited toReichardt's dyes, 4-hydroxystyryl-pyridinium dye,4-methoxycarbonyl-1-ethylpyridinium iodide, and2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)-phenolate.

The addition of at least one Brønsted acid dye to an array containing atleast one metal ion-containing Lewis acid dye can improve thesensitivity of the array for particular analytes and increase theability to discriminate between analytes. For example, a colorimetricsensing element similar to that of the present invention has been shownto detect volatile organic compounds and complex mixtures down to ppblevels (Rakow, et al. (2005) Angew. Chem. Int. Ed. 44:4528-4532).Further, the use of one or more metal ion-containing dyes in combinationwith one or more Brønsted acid dyes can advantageously create asignature indicative of the presence of a particular analyte. Thus,while some embodiments embrace the use of at least one Lewis acid and/orbase dye, one Brønsted acidic and/or basic dye, or one zwitterionicsolvatochromic dye, other embodiments of this invention embrace the useat least two different classes of dyes on the instant arrays. In oneembodiment, the colorimetric sensing element contains at least one Lewisacid and/or base dye, one Brønsted acidic and/or basic dye, or onezwitterionic solvatochromic dye. In another embodiment, the colorimetricsensing element contains at least one Lewis acid and/or base dye and oneBrønsted acidic and/or basic dye. In a further embodiment, thecolorimetric sensing element contains at least one Lewis acid and/orbase dye and one zwitterionic solvatochromic dye. In yet a furtherembodiment, the colorimetric sensing element contains at least oneBrønsted acidic and/or basic dye and one zwitterionic solvatochromicdye. Still further embodiments embrace the use at least three differentclasses of dyes on the instant arrays, i.e., at least one Lewis acidand/or base dye, one Brønsted acidic and/or basic dye, and onezwitterionic solvatochromic dye.

The interference of atmospheric humidity on sensor performance is aproblem with cross-responsive sensors of the prior art. The highconcentration of water vapor in the environment and its large andchangeable range makes the accurate detection of analytes at lowconcentration difficult with the prior art sensors. Water vapor rangesin the environment from <2000 to >20,000 ppmv. Thus, when detecting afew ppmv of an analyte, or even a few ppb, even a very low level ofinterference from water is intolerable. Physisorption of molecules onsurfaces is dominated by the relative hydrophobicity of the adsorbateand adsorbent. Therefore, a disadvantage of the cross-responsive sensortechnology of the prior art is sensitivity to changes in humidity.

In contrast, the dyes of the instant colorimetric sensing element areselected from hydrophobic, water-insoluble dyes which arecontact-printed as non-aqueous, hydrophobic solutions onto hydrophobicsubstrates. As such, the instant colorimetric sensing element isessentially impervious to changes in relative humidity (RH). Forexample, a colorimetric sensing element exposed to water vapor from purewater (RH 100%) or to saturated salt solutions whose water vaporpressures ranged from 11% to 94% RH shows that the dyes in thecolorimetric sensing element are unresponsive to water vapor. Similarly,the response to other analytes is not affected by the presence orabsence of RH over this range. As such, particular embodiments of theinstant colorimetric sensing elements can be used directly in water forthe sensing of dilute aqueous solutions of organic compounds (Zhang &Suslick (2005) supra). Therefore, in particular embodiments,chemoresponsive dyes of the instant invention are hydrophobic orwater-insoluble. As used herein hydrophobic is used in the conventionalsense to describe a compound which is incapable of dissolving in water.

Advantageously, the instant colorimetric sensing element probes the fullrange of intermolecular interactions to facilitate the detection ofanalytes such as amines, phosphines, thiols, alcohol, etc., produced bymicroorganisms. By way of illustration Table 1 provides a list of dyes,the analytes which the dyes can detect, and the resulting color change.Further, the sensing element of the invention is sensitive and robust(i.e., stable to exposure to analytes or the environment). Desirably,this is achieved by employing disposable sensors, which are notintegrated to the readout device, thus unlinking the opposing demands ofthe sensor.

TABLE 1 Dye Analyte Color Change Cresol Red (basic) Carbon dioxideViolet −> Yellow Phenol Red (basic) Carbon dioxide Red −> YellowBromocresol Green Ammonia Yellow −> Green Yellow −> Blue Reichardt's DyeAcetic Acid Blue −> Colorless Tetraphenylporphirinato Ethanol Green −>Brown manganese (III) chloride [MnTPPCl] TetraphenylporphirinatoPyridine Red −> Green cobalt (III) chloride [CoTPPCl] Zinctetraphenylorphyrin Methyl amine Maroon −> Brown [ZnTPP]Tetraphenylporphyrin Hydrogen Brown −> Green [H₂TPP] chlorideTetraphenylporphyrin Ammonia Green −> Brown [H₄ ⁺²TPP] (diprotonated)Bismuth (III) Hydrogen Sulfide Colorless −> neodecanoate BlackTetra(2,6- Hydrogen Cyanide Brown −> Green dihydroxy)phenylporphyrin(with HgBr₂) Copper (II) Hydrogen Sulfide Sky blue −>acetylacetonate Brown Copper (II) Methanethiol Sky blue −>acetylacetonate Brown Palladium(II) acetate Methanethiol Light yellow −>Dark Yellow Palladium(II) acetate Hydrogen Sulfide Light yellow −> BrownZinc Chlorine Deep pink −> tetramesitylporphyrin Green (ZnTMP) ThymolBlue Triethyl amine Maroon −> Brown Zinc Tetra(2,6- Alcohol Pink −>Sandy difluorophenyl)porphyrin brown

The present invention is an improvement over the “optoelectronic nose”which is based on the colorimetric array detection using a chemicallydiverse range of chemically responsive dyes (Rakow & Suslick (2000)supra; Suslick & Rakow (2001) supra; Suslick, et al. (2004) supra;Suslick (2004) supra; Rakow, et al. (2005) supra; Zhang & Suslick (2005)supra; U.S. Pat. Nos. 6,368,558 and 6,495,102). In the instantinvention, olfactory-like responses are converted to a visual outputwhich can be readily detected and analyzed by digital imaging andpattern recognition techniques (Beebe, et al. (1998) Chemometrics:Practical Guide; J. Wiley & Sons, Inc.: New York; Haswell, Ed. (1992)Practical Guide to Chemometrics; Marcel Dekker, Inc.: New York).

In this regard, the apparatus of the instant invention can further becombined with a visual imaging means for monitoring changes of thesensing element. In embodiments pertaining to a colorimetric sensingelement, the visual imaging means monitors the spectroscopic response,transmission response or reflectance response of the dyes on thecolorimetric sensing element at one or more wavelengths in a spatiallyresolved fashion so that all of the spots in the colorimetric sensingelement are individually imaged or addressed and the color of each spotis individually determined. For the purposes of the present invention,the terms color and colorimetric are intended to include wavelengths inthe visible portion of the electromagnetic spectrum, as well as theinvisible portion of the electromagnetic spectrum, e.g., infrared andultraviolet. Color detection can be accomplished with an imagingspectrophotometer, a flatbed scanner, slide scanner, a video or CCD orCMOS digital camera, or a light source combined with a CCD or CMOSdetector. Any still or video as well as analog or digital camera can beemployed. Moreover, any imaging format can be used, e.g., RGB (red,green and blue) or YUV. Even the simple gray scale imaging can be used.When used in combination with colorimetric sensing elements and imageanalysis software, colorimetric differences can be generated bysubtracting the RGB values of dye images generated before and afterexposure of the dye to a sample. The colorimetric differences representhue and intensity profiles for the array in response to analytesproduced by microorganisms. This eliminates the need for extensive andexpensive signal transduction hardware associated with previoustechniques (e.g., piezoelectric or semiconductor sensors). When used inaccordance with the method of the present invention, a unique colorchange signature can be created which provides both qualitativerecognition and quantitative analysis of microorganisms present in asample.

The sensitivity of the instant colorimetric sensing element is primarilya function of two factors, the ability of a dye spot to change colorwhen exposed to an analyte and the ability of the imaging system todetect that color change. An optical spectroscopic measurement systemcan divide the visible spectrum into as many as 500 individual bandpasswindows whereas a three-color imaging system by definition contains onlythree such windows. An optical spectroscopic measurement system istherefore capable of detecting smaller color changes than can bedetected by three-color imaging systems, effectively increasing thesensitivity of the entire cross-responsive sensing system. Accordingly,in particular embodiments of the present invention, an opticalspectroscopic measurement system is employed as a visual imaging means.As used herein, optical spectroscopic measurement systems refer to anysystem that yields higher color resolution than a three-color imagingsystem. This can be an imaging spectrograph, fiber optic probe(s)coupled to a spectrograph, or other spectroscopic system.

As shown in FIG. 6, the instant apparatus can be a component of anintegrated system 100 in which a visual imaging means 110 (e.g., ascanner) is housed in an incubator or growth chamber 120 to maintain themicroorganisms at a particular temperature. In the system 100 depictedin FIG. 6A, retractable rack 130 holds apparatus 10, wherein rack 130can extend when apparatus 10 is to be loaded and retract to a restingposition on the bed of the visual imaging means 110 after loading.System 100 can further contain aerating means 140 to agitate apparatus10, wherein lifting means 150 raises and lowers aerating means 140 toposition apparatus 10 adjacent to visual imaging means 110 (see FIG.6B). Aerating means 140 can be a conventional rocker or shaker whichagitates apparatus 10 at low amplitude and frequency to keep thecontents therein aerated and homogeneous. System 100 can be driven bysoftware on computer 160, which would register the time at which eachapparatus 10 was loaded, drive visual imaging means 110 and process theimages, perform data analysis on the color changes occurring duringincubation, and output answers such as presence or absence of particularmicroorganisms and, when particular microorganism are present in thesample, identify the species and strain.

To provide data analysis, the instant apparatus can be combined withstandard chemometric statistical analyses (e.g., principal componentanalysis, hierarchal cluster analysis, and linear discriminantanalysis), an artificial neural network (ANN), or other patternrecognition algorithms to correlate dye color changes to variousanalytes and bacteria species.

There is extensive classification information in the temporal or kineticresponse of individual dyes as microorganisms grow and undergo changesin metabolism. An example of this behavior is shown in FIG. 7, whichcharts the color changes of several dye spots exposed to the headspaceof a growing culture of microorganisms. Several dye spots undergoreversible color change and the rate of response varies for differentdyes. This pattern is different and unique for each species and strainof microorganism.

These temporal color changes can be analyzed using principal componentanalysis (PCA) to provide bacteria classification and/or identification.PCA determines the number of meaningful, independent dimensions probedby an apparatus of the invention and creates a new coordinate spacedefined by these dimensions. This space is referred to as “PCA space.”Each bacteria species is represented by coordinates in PCA space.Vectors from incoming samples, i.e., unknown microorganisms, areprojected onto this new coordinate space and the distance between theunknown vector and the various microorganism vectors in the training setare calculated. The result is a numerical “probability ofclassification” as each type of microorganism used to build the trainingset.

The instant array probes a much wider range of chemical interactionsthan do prior art array sensors, thus the dispersion of the instantcolorimetric sensing element is increased over prior art sensingelements. It is this increased dimensionality that permits thediscrimination among very closely related compounds, e.g., decylamineversus undecylamine in PCA space.

In hierarchical cluster analysis, the three color channels correspondingto each dye channel can be thought of as vectors in n-dimensional space,where n=3*N (3 color channels per each of N spots). Hierarchical clusteranalysis on the composite n-dimensional vectors at either a single timepoint or “time stacked” over multiple time points partitions the datainto clusters, with each cluster containing all samples of a givenmicroorganism species without cross-classification. As shown in FIG. 7,several species of microorganisms were successfully classified usinghierarchical cluster analysis.

A third method, linear discriminant analysis, operates on a training setof data to define a new n-dimensional vector space in which thecoordinates are selected so as to minimize the distance between matchingvectors (same microorganism species) and maximize the distance betweendissimilar vectors (different microorganisms species). Vectors fromincoming samples, i.e., unknown microorganisms, are projected onto thisnew coordinate space and the distance between unknown vector and thevarious microorganism vectors in the training set are calculated. Theresult is a numerical “probability of classification” as each type ofmicroorganism used to build the training set. An example of lineardiscriminant analysis used to classify different strains of E. coli isshown in FIG. 8.

By way of further illustration, ANN is an information processing systemthat functions similar to the way the brain and nervous system processinformation (Tuang, et al. (1999) FEMS Microbiol. Lett. 177: 249-256).The ANN is trained for the analysis and then tested to validate themethod. In the training process, the ANN is configured for patternrecognition, data classification, and forecasting. Commercial softwareprograms are available for this type of data analysis. To illustrate, astandardized set of data from an array of dyes exposed to ammonia servesas the input vector. The desired output vector is the classification ofammonia (e.g., produced by Helicobacter), “0” for no ammonia (i.e., noHelicobacter) and “1” for ammonia (i.e., Helicobacter). Training isaccomplished by using the standardized data set and associating theinput or signature with the desired output or classification. Theprogram compares the data and computes network output with the desiredoutput until an acceptable level of recognition is achieved.

Using such analysis, the instant apparatus can be used for detecting andidentifying any microorganism for the purposes of, e.g., diagnosing aninfection or detecting a microbial contaminant. Bacterial infectionsemit specific analytes. The classic diagnosis of strep throat was madeby smelling the breath of the patient. Many disease states areassociated with distinctive aromas, and the detection of biomarkersrepresents a fundamental window on the internal functioning of the body(Pavlou, et al. (2000) Biosensors & Bioelectronics 15:333-342). Thefield of gas chromatography has extensively studied the volatilechemicals emitted from microorganisms, and it is clear that differentspecies, even strains with small genetic differences, emit a distinctprofile of enzymatic products in the form of volatile organic compoundssuch as amines, sulfides, and fatty acids (Zechman & Labows (1984) Can.J. Microbiol. 31:232-237). The analytes emitted from variousmicroorganisms have been studied in some detail. For example, E. coliemits acetic acid when grown in a glucose rich media and ammonia andamines in a protein rich media. Pseudomonads produce alcohols, ketones,and amines, whereas S. aureus emits amines, sulfides, and alkenes. Theseanalytes can accumulate to substantial levels, allowing for detectionwith a cross-responsive sensor in the headspace above the microorganismsor within the growth media itself.

The instant apparatus utilizes this fact to detect and identify severalbacterial species, including Escherichia coli, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pyogenes, Staphylococcusepidermidis, Staphylococcus sciuri, and Moraxella (Brauhamella)catarrhalis.

The instant apparatus is useful over the prior art in that changes inthe colorimetric sensing element due to microbial metabolism (e.g.,increases or decreases in a gaseous compound due to metabolism) aredetected in the atmosphere over the growth medium or dissolved in thegrowth medium; measurements can be made from outside the transparentwall of the container without having to violate the integrity of thecontainer because the sensor is affixed to the interior surface of thecontainer. The external measurements can be made by visual inspection orwith an instrument that measures spectroscopic, transmission orreflectance responses, or by image capture; opaque/colored orfluorescent components in the specimen do not interfere with the abilityto detect changes or the measurement of those changes; and a pluralityof chemoresponsive dyes provides the detection, quantitation, andidentification of one or more microorganisms present in the specimen.

Given such advantages, the present invention is also a method fordetecting, quantifying and/or identifying a microorganism in a sample.The method involves depositing a sample suspected of containing amicroorganism on the medium of the apparatus of the present inventionand monitoring changes of the sensing element. In some embodiments,changes include spectroscopic, transmission or reflectance responses ofthe plurality of chemoresponsive dyes, wherein the detected responsesare indicative of the presence and identity of the microorganism.

The sample suspected of containing a microorganism can be a fluid,semi-fluid, or solid sample from a biological or environmental source.Sample can include, e.g., a food item, blood, semen, sputum, mucous,feces, or other bodily discharge as well as a soil or water sample. Inthe instant method, the sample can be introduced onto or into the mediumusing any conventional means including spreading the sample across themedium with a glass rod or inoculation loop and injection of liquid witha needle and syringe. The apparatus is then incubated, promoting thegrowth of microorganism colonies. Dyes of the colorimetric sensingelement located in the medium or the headspace of the microorganismsundergo detectable changes and, upon manual or automatic inspection ofthe array, the presence and identity of microorganism(s) in the sampleis determined.

If the colorimetric sensing element is inspected automatically, a systemis provided which performs three main functions: incubation of theapparatus, image acquisition/capture, and image processing. The systemprovides a controlled environment for incubating and promoting growth ofmicroorganisms, which can include a heater if incubation is to takeplace at an elevated temperature from ambient (though an elevatedtemperature is not necessary in all situations). Upon adding the sampleto the medium of the apparatus, the apparatus is placed in the incubatorwhere it is subsequently sensed/observed by a visual imaging means forimage acquisition/capture (e.g., a camera or scanner) during theincubation period. Images of the colorimetric sensing element can becaptured at regular predetermined intervals and subsequently analyzedusing one or more image processing techniques and algorithms todetermine the presence and identity of one or more microorganisms on orin the medium.

As will be appreciated by the skilled artisan, the apparatus and themethod of the instant invention can be employed in the detection andidentification of a variety of microorganisms. Identification can be onthe basis of whether a bacterium is Gram positive or Gram negative, oralternatively, identification of particular genera, species or strainscan be achieved. The instant apparatus and method finds application inthe detection of the presence or absence of numerous bacteria including,but not limited to, Bacillus anthracia, Bordetella pertussis,Clostridium botulinum, C. tetani, Corynebacterium diphtherias,Escherichia coli, Moraxella (Brauhamella) catarrhalis, Pseudomonasaeruginosa, Shigella spp., Staphylococcus aureus, Streptococcuspneumoniae, S. pyogenes, and Vibrio cholerae; fungi such as Microsporumsp., Trichophyton sp., Epidermophyton sp., Sporothrix schenckii,Wangiella dermatitidis, Pseudallescheria boydii, Madurella grisea,Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis,Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus niger, andCandida albicans as well as protozoa and other parasitic microorganisms.Beneficial and detrimental microorganisms from food, soil, or watersamples can also be detected, quantified and identified.

The instant apparatus can be provided to a user in a kit. For example,in embodiments pertaining to a Petri dish-type apparatus, the kit caninclude the bottom of the Petri dish containing the colorimetric sensingelement and retainer means already in place with the entire assembly ina hermetic pouch. The other half of the Petri dish containing the mediumcan be in a separate pouch. When the device is to be used to detect,quantify or identify one or more microorganisms in a sample, the samplein question is deposited or smeared on the medium and the two halves ofthe apparatus are brought together or otherwise sealed to form theassembled apparatus seen in FIGS. 1B and 1C. The sealed apparatus isthen placed on an imaging platform for monitoring and analysis. Inembodiments pertaining to a bottle-shaped configuration, the kit caninclude the bottle with the sensing element and medium already in thebottle. The kit of the invention can also contain other components suchas sterile water for diluting the sample, instructions for using theapparatus as well as controls (e.g., a sample of a known concentrationof a particular microorganism) and examples of array data indicative ofa particular microorganism.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Materials

With the exception of the colorimetric sensing elements, all othermaterials were obtained from the following sources. BACTO™ tryptic soybroth and columbia anaerobic sheep blood agar plates were purchased fromFisher Scientific, Inc (Hampton, N.H.). Sheep serum (S2263) waspurchased from Sigma-Aldrich (St. Louis, Mo.). The type of media usedfor these experiments was chosen because it contains a food source forall of the bacteria studied and all of the species were known to grow onblood agar. Also, it was desirable to develop a system that could beused universally for sensing of all types of volatile bacterialmetabolites. Bacteria were purchased from American Type CultureCollection (ATCC, (Manassas, Va.). All materials were used as receivedand bacteria cultures were stored at −70° C. until used. All UV-vismeasurements were carried out at 600 nm against an appropriate blank.

Escherichia coli is a gram-negative bacterium that grows on a variety ofagar media with an optimal temperature range between 30° C. and 37° C.Colonies on nutrient or blood agar are cream to tan in color, circular,and smooth. The bacterium catabolizes glucose and other carbohydratesfound in the media to form acids and “gases” (Wistreich (1999)Microbiology Perspectives, A Photographic Survey of the Microbial World;Prentice Hall: Upper Saddle River, N.J.). The most common diseasescaused by E. coli are gastroenteritis, diarrhea, and urinary tractinfections. Isolation and biochemical testing are the conventionalapproaches used for identification. In particular, specific, selective,and differential media and immunologic tests such as the particleagglutination procedure are conventionally used for the identificationof E. coli O157:H7 strains.

Pseudomonas aeruginosa is a gram-negative, Bio Safety Level II bacteriumthat grows best at 37° C. It oxidizes glucose to carboxylic acids butdoes not produce acid from disaccharides such as lactose. Colonies arelarge and translucent with irregular edges, and usually appear within 24to 48 hours. P. aeruginosa is an opportunistic pathogen which colonizesand invades injured epithelial surfaces. 75% of all intensive care unitspatients are colonized by this pathogen. Common diseases caused by thispathogen include pneumonia, chronic respiratory infections, andbacterial meningitis. Treatment of P. aeruginosa is difficult due to itsresistance to antibiotics. Most strains of P. aeruginosa areconventionally identified on the basis of their characteristicgrape-like odor, colonial morphology, and the production of awater-soluble blue pigment, pyocyanin (Campa, et al, Ed. (1993)Pseudomonas aeruginosa as an Opportunistic Pathogen; Plenum Press: NewYork).

Staphylococcus aureus is a gram-positive, Bio Safety Level II bacteriumwhich grows best between 30° C. and 37° C. Colonies are usually opaque,smooth, circular, and yellow or sometimes yellow-orange. S. aureus iscatalase positive and oxidase negative. Several key characteristics ofS. aureus have been conventionally used to identify the organism anddistinguish it from others. These include fermentation of mannitol onmannitol-salt agar, DNAse production, and a positive coagulase test. Onaverage 33% of healthy individuals carry S. aureus (A. L. Honeyman &Bendinelli, Ed. (2001) Staphylococcus aureus Infection and Disease;Kluwer Academic/Plenum Publishers: New York). This bacterium is known tocause endocarditis, meningitis, pneumonia, and septic arthritis.Commercially available molecular probes and identification systems,immunological tests, and bacterial viruses are used for detection andidentification.

Streptococcus pyogenes is a gram-positive, Bio Safety Level II bacteriumwhich grows optimally at 37° C. S. pyogenes is associated with group Astreptococci (GAS) along with Streptococcus pneumoniae. Common diseasescaused by S. pyogenes include strep throat, scarlet fever, pneumonia,and septicemia. Traditional diagnosis relies on a throat culture, inwhich the bacteria appear as beta-hemolytic colonies on 5% sheep bloodagar after 24 to 48 hours.⁴¹

Moraxella (Brauhamella) catarrhalis is a gram-negative, Bio Safety LevelII bacterium that grows on nutrient and blood agar to yield small,circular, convex colonies which are usually grayish white. M.catarrhalis exhibits optimal growth between 33° C. to 35° C. M.catarrhalis is catalase and oxidase positive and does not produce acidfrom carbohydrates. This pathogen commonly causes bronchitis, pneumonia,sinusitis, and meningitis. Diagnosis is conventionally obtained byisolation and typical biochemical testing.

Example 2 Preparation of Liquid Media

Tryptic soy broth was prepared by dissolving 30 grams of the powderedmedia into 1 L of purified water. The resulting mixture was divided into250 mL aliquots and autoclaved at 121° C. for 15 minutes. The bottleswere then cooled to room temperature and an aliquot of ˜12.5 mL ofsheep's serum (prepared beforehand and stored at −70° C. until needed)was added to create an approximate 5% sheep's serum solution by volume.Media bottles that were not used immediately were stored at roomtemperature without the addition of sheep serum.

Example 3 Growth of Bacteria on Solid Media

Before each experiment, a solid media streak plate was prepared fromfrozen permanent cultures according to conventional methods (Freund &Lewis (1995) Proc. Natl. Acad. Sci. USA FIELD Publication Date: 1995Mar. 28 92:2652-2656) to obtain single colonies for growth in liquidmedia culture. All plates were incubated at 37° C. until colonies werevisible. Streak plates were then wrapped in PARAFILM and stored at 4° C.until further use. Each plate was used for no longer than one month toensure colony viability, with the following exceptions. Culturescontaining M. catarrhalis or S. pyogenes were prepared weekly becausethe relevant growth of these organisms on blood agar was less robustthan that of other species being tested.

All solid media experiments were conducted at 37° C. in the incubatorwith a flatbed scanner housed therein. Colorimetric sensing elementswere exposed to volatile bacterial metabolites by placing them printside down into the bottom of the appropriate agar plate inoculated witha desired bacterial species. Petri dishes were placed on a flatbedscanner, and images were collected through the clear plastic of thepetri dish using CHEMSCAN software.

Two configurations of plates were analyzed. In the first configuration,each 100-mm diameter petri dish contained four individual colorimetricsensing elements and the scanner held six dishes. In the secondconfiguration, 60-mm petri dishes were used which held one colorimetricsensing element and the scanner held twelve petri dishes. To rule outbackground noise from the response of the agar, the signal arising frominoculated agar at 30 minutes was subtracted from all subsequent timepoints. Multiple experiments with uninoculated agar revealed that noresponse to the agar alone was observed after 30 minutes. In addition,control experiments were conducted in which the agar was inoculated withliquid media that contained no bacteria. No signal above that of theagar plate was detected for uninoculated liquid media.

Volatile bacterial metabolite experiments were conducted for at least a12-hour period with data acquisition occurring at least every 30minutes. Color change values for the colorimetric sensing elements forall experiments were determined using a customized software package,CHEMEYE™ (ChemSensing, Inc.). Digital data was compiled into a databasecontaining each trial run. Chemometric analysis of this database wasperformed using commercially available software.

The following steps were taken to prepare bacterial species for eachexperiment. Approximately 5 mL of the appropriate liquid media wasinoculated with a single colony of a desired bacteria species from thestreak plates. The liquid culture was allowed to grow overnight in awater bath shaker at 37° C. The optical density of the culture wassubsequently measured and the solution was diluted to the desiredoptical density (depending on the growth rate of the species) in either5 or 10 mL of fresh liquid media. The bacteria were then allowed to growuntil the start of the experiments (usually 4 hours). A final opticaldensity measurement was made immediately prior to the start of thevolatile bacterial metabolite sensing experiment, the concentration wasadjusted a final time, and an appropriate volume of the bacterialculture was spread onto the appropriate agar plate for exposure tocolorimetric sensing elements.

Colorimetric sensing elements in inoculated and sealed petri dishes wereimaged at regular time intervals post-inoculation. The imaging systememployed possessed the ability to collect spatially resolved data sothat all of the spots in the colorimetric sensing elements could beindividually imaged or addressed and the color of each spot could beindividually determined.

Example 4 Limits of Detection

For the purposes of the instant apparatus, limit of detection is definedas the smallest number of bacteria that can be detected by colorimetricsensing elements positioned in the headspace of the bacteria. The limitof detection was studied by inoculating tryptic soy agar in petri disheswith a range of colony forming units (cfu) of E. coli. The petri dishwas then closed with the colorimetric sensing element in the headspaceof the growing bacteria, and the color of the colorimetric sensingelement was monitored throughout the growth of the bacteria. The numberof colony forming units for each inoculation was determined by countingthe visible colonies on each plate at the end of the sensing experiment.Multiple experiments were performed at cfu loadings from 5 cfu to 10⁵cfu. A typical colorimetric sensing element response pattern and thetime needed to detect representative cfu loadings are shown in FIG. 9.

FIG. 10 shows that time needed to detect an inoculum of 1020 cfu E. coliis approximately 480 minutes (as shown in FIG. 9). The color changecurves resulting from lower bacterial loadings contain similar featuresat and after the time of detection and differ only in the duration oftime needed to reach that point. One of the lowest detectable cfu countsmeasured was 5 cfu, wherein this loading gave detectable signal at 930minutes.

Example 5 Monitoring Bacterial Metabolism

Many bacteria are known to follow several metabolic pathways to yieldspecific metabolites. When presented with a variety of media sources,most bacteria will consume all of one food type then continue on to thenext. For example, when presented with a mixture of glucose and lactose,E. coli will consume the glucose first, and subsequently consume thelactose only when all the glucose is gone.

To demonstrate that a colorimetric sensing element can detect theseshifts, the change in RGB values was plotted as a function of time forhigh loadings of bacteria growing on tryptic soy agar with 5% sheepblood. The colorimetric sensing element allows for the monitoring of thegrowth stage as well as any changes in emitted metabolites duringgrowth. Therefore, changes in RGB were monitored for E. coli, P.aeruginosa, S. aureus, S. pyogenes, and M. catarrhalis as a function oftime. All graphs appeared somewhat similar with large changes in RGBoccurring at the ˜270 minute mark. Some pronounced shifts were observedin both the E. coli and M. catarrhalis plots, in which some RGB valuesinitially became more negative, and then at ˜420 minutes increasedagain. It is believed that this behavior was caused by the depletion ofglucose in the medium and subsequent switch to protein as a source ofnutrients. Initial colorimetric sensing element responses correspondedto the production of acid from the growing bacteria. Upon cessation ofacidification, a reversal in color change direction was observed. Thismay correspond to the production of basic analytes, such as ammonia andvarious amines, as the protein in the medium was metabolized. Similarshifts were seen for several other bacteria, to include S. aureus and S.pyogenes.

Example 6 Solid Media Growth Curves

The number of bacteria present and producing volatile metabolites at anygiven time is directly related to how quickly the bacteria double. Sincebacteria grow exponentially, the number of bacteria present after aspecific growth time is dependent on the initial number of bacteriapresent in the culture. Each new generation of cells produced leads todouble the previous amount. The equation to determine the number ofbacteria present at any given time during growth is given below:

∫_(t_(i))^(t_(f))tN_(o)2^(i/t)t

where t_(f) is the final time, t_(i) is the initial time, t is time,N_(o) is initial number of bacteria and t_(d) is doubling time. Thedoubling time (t_(d)) is dependent on the bacteria and the media source,as well as the environmental conditions.

Conventional growth curves are determined by measuring the opticaldensity of bacteria grown in liquid culture. However, there is no directmethod of obtaining growth curves of bacteria grown on solid media.Traditional methods for monitoring growth of bacteria grown on solidmedia require the use a special soft agar. An initial amount ofbacteria, determined by measuring the optical density, is plated as alawn and allowed to grow for a desired period of time, upon which thebacteria are scraped off the plate and dissolved in fresh liquid medium.UV/VIS spectroscopy is then performed to determine O.D. and the numberof bacteria present is then back calculated. A separate agar plate isrequired for each time point used in determining the growth curves. Assuch, this method is time consuming and prone to error and humanvariability.

Advantageously, the response of colorimetric sensing elements to growingbacterial cultures is an initial sharp increase in signal and then,after a time that varies with the species being monitored, reaches agenerally constant level. A plot of this change with time yields agrowth curve. The change in colorimetric sensing element response isdefined as the total minimum variance which is the normalized Euclideandistance of all 108 RGB values for each time point examined duringbacterial growth. In other words, it is a measure of distance betweentime points in 108-dimensional space for a colorimetric sensing elementthat contains 36 spots. Using changes in colorimetric sensing elementresponse to monitor growth of E. coli on tryptic soy agar with 5% sheepblood, it was found that the growth curve observed on solid mediaexhibits features found in conventional liquid growth curves; a lagphase followed by an exponential growth phase and a stationary phase.

All bacteria studied have similar growth curves when grown on the samesolid media. As shown in FIG. 11, the general shape of the curve is thesame for all bacteria tested, with growth rate being species-dependent.These growth curves resemble those corresponding to growth in liquidmedia.

Example 7 Colorimetric Sensing Elements and Liquid/Blood Culturing

Sepsis is typically associated with blood bacteria concentrations ofless than 10 colony forming units (cfu) per mL of blood. Thisconcentration does not readily allow for the identification of thebacteria using non-DNA based techniques. As such, the bacteria aregenerally grown, or cultured, to provide a sufficient number of bacteriafor identification.

The blood culturing process involves obtaining blood samples from thepatient suspected of being septic, and injecting each sample into adedicated blood culture bottle, which contains a liquid medium thatsupports the growth of the bacteria, a gas tight seal that preventsanalytes generated by the growing bacteria from escaping and allowingfor pressure to develop in the bottle, and a sensor that indicateswhether or not bacteria are present. Blood culture bottles are typicallyinterfaced to a monitoring incubator that maintains a temperature of 37°C. and reads the sensor to alert the user when the presence of bacteriahas been verified. A bottle that does not register positive for bacteriawithin 5 days is typically considered to be negative for bacteria.

Conventional blood culturing sensing systems employ a CO₂ sensor, eithercolorimetric or fluorescent in nature, or a pressure sensor. However,CO₂ is inherently only weakly reactive relative to many other gases andthus greater concentrations of CO₂ are required to register a change incolor or fluorescence. By employing the instant colorimetric sensingelement in a blood/liquid culture system, more reactive analytes can beand are detected, thereby decreasing the time needed to detect bacteriain a blood culture bottle. Moreover, whereas conventional blood culturesystems are designed only for bacterial detection, the instantcolorimetric sensing element can also readily identify the species ofbacteria based on the analytes evolved during growth. In particular, theinstant colorimetric sensing element is sensitive to bacterial analytessuch as amines and carboxylic acids at sub part per million (ppm)levels; sulfides and thiols on ppm levels; CO₂ at sub ten ppm levels;and aldehydes and alcohols, though at higher concentrations.

To demonstrate the use of the instant apparatus in blood culturing, E.coli was grown in liquid media overnight and diluted to the desiredoptical density prior to use. A mixture of 20% human blood and 80%liquid media were then added to the container, in this case a bottle,followed by the appropriate volume of the bacterial solution to attainthe desired initial number of bacteria in the blood culture bottle.Blood was freshly drawn from human subjects and used within 10 minutesafter draw. Subsequently, the cap was placed on the bottle, the bottleloaded onto a shaking and imagining platform, and the zero-point orbaseline image collected. After several experiments, it was observedthat a signal arises from the media itself but this signal reached asteady state after thirty minutes. As such, all blood culture data ispresented with the thirty minute point serving as the baseline.

FIG. 12 shows representative results from a single blood cultureexperiment. Each colored line in the graph represents a single colorchannel, where “color channel” is defined as the individual Red, Greenand Blue components of each dye spot. For instance, the red component ofdye spot 12 is termed color channel R12, the green component of dye spot12 is color channel G12, etc. Definite signal-above-baseline wasobservable by the three-hour point.

To examine the signal arising from exposure of the colorimetric sensingelement to liquid medium only, the colorimetric sensing element wasexposed to pure medium in the absence of blood and bacteria. In threeseparate experiments, which monitored colorimetric changes over aseven-hour period, all colorimetric signal array changes were in therange of −20 to +20, with little fluctuation in signal throughout thecourse of the experiment.

Given that human blood cells can remain vital and produce analytes whenincubated in liquid medium of the sort used herein, colorimetric signalarray changes arising from human blood and medium alone were determinedin the absence of bacteria. The results from nine separate experimentsindicated that the signal arising from blood/media mixtures, while insome cases larger than that arising from pure media alone, did notinterfere with the detection of bacteria.

The detection of E. coli growing in 20% human blood and 80% medium wassubsequently analyzed. Colorimetric sensing element signal resultingfrom “high” loadings of E. coli in the blood/media mixture wasdetermined. High loadings were defined as an initial concentration inthe blood culture bottle of at least 10⁴ cfu/mL as determined by opticaldensity measurements of the liquid bacterial culture prior toinoculating the blood/media. Separate experiments were conducted withmedia which support aerobic microorganisms and anaerobic microorganisms.While the experiments were not conducted under anaerobic conditions, aclear difference between the signals arising from bacteria grown inthese two media was apparent. In all cases the bacteria was easilydetected within three hours after inoculation of the blood/mediamixture.

In addition to “high” loading, colorimetric sensing element signalresulting from “low” loadings of E. coli was also determined. Lowloadings were defined as an initial cell concentrations ranging from 500cfu/mL to 4000 cfu/mL. The results of this analysis indicated that inall cases the signal was substantially stronger than that observed fromblood/media alone. Furthermore, signal from low loadings differed fromthat of high loadings in two ways; longer time for detection and adecrease in the negative spike. The data at low loadings of E. colirepresented a close approximation of an expanded version of the dataobtained with high loadings of E. coli in which similar featuresoccurred but at later times.

To define the detection of bacteria over signal from blood/media, amathematical formalism was developed. In other words, a rigorous methodof analyzing the colorimetric sensing element signal was created toyield a yes or no answer that did not depend upon visual analysis of agraph. Analysis of blood/media as compared to low load or high loadcultures was conducted. In this analysis, the signal from blood/mediaresembled that of a low load culture and it was difficult to discern byvisual examination of the graphs whether the low load culture containedbacteria or not (the initial concentration of cells in low load was 200cfu/mL as measured by optical density followed by serial dilutions). Assuch, to definitively determine the presence of bacteria in the low loadculture, a closer examination of color channels G22 and B28 was carriedout, as G22 and B28 exhibited a steady upward increase in several of theblood/media instances but showed structure in the case of samplesinoculated with bacteria. This structure was most apparent when thesecond derivative of each of the two lines was plotted as a function oftime (FIG. 13). The second derivatives were calculated by first fittingeach line to a sigmoid, a twice continuously differentiable functionthat closely approximates the actual data. The second derivative of“media” (the blood/media mixture) was essentially flat and never reacheda value greater than ±0.0003. In contrast to the flat nature of theblood/media curve, all other samples exhibited a second derivativegreater than ±0.003, an order of magnitude greater (FIG. 13).

To further demonstrate that the apparatus is applicable to theidentification of bacteria growing in liquid media, a colorimetricsensing element was placed in bottles inoculated with bacteria loadingscomparable to those observed in clinical samples: between 20 and 80 cfuper bottle. This corresponds to 2 to 8 cfu/mL in blood drawn from aseptic patient. As shown in FIG. 14, each of the bacteria (i.e., E coli,S. aureus, E. faecalis, and P. aeruginosa) generated a uniquecolorimetric sensing element response pattern that clearly identifiedthe bacteria.

What is claimed is:
 1. An apparatus for detecting, quantifying oridentifying a microorganism comprising (a) a container with at least onechamber; (b) a medium within the chamber of the container, wherein saidmedium is supplemented with nutrients for supporting growth of amicroorganism; and (c) at least one colorimetric sensing element placedin, or proximate to, the medium, wherein said sensing element includes asubstrate comprising a plurality of chemoresponsive dyes in apredetermined pattern combination, wherein monitoring spectroscopic,transmission or reflectance responses of the plurality ofchemoresponsive dyes is used to detect, quantify, or identify amicroorganism.
 2. The apparatus of claim 1, wherein the container isbottle or vial as a container.
 3. The apparatus of claim 1, wherein thecontainer is sealed or sealable.
 4. The apparatus of claim 1, whereinthe container comprises an overlapping radial seal or a capping seal. 5.The apparatus of claim 1, wherein the medium is a liquid, semi-liquid,semi-solid or solid medium.
 6. The apparatus of claim 1, wherein thesubstrate is a chromatography plate, paper, filter paper, porousmembrane, polymer, glass, metal or hydrophobic substrate.
 7. Theapparatus of claim 1, wherein the plurality of chemoresponsive dyes areprinted on the substrate by airbrushing, ink-jet printing, screenprinting, stamping, micropipette spotting, or nanoliter dispensing. 8.The apparatus of claim 1, wherein the substrate is opaque.
 9. A systemcomprising the apparatus of claim 1 and a retractable rack for holdingthe apparatus.
 10. The system of claim 9, wherein the colorimetricsensing element is automatically inspected.
 11. The system of claim 9,wherein spectroscopic, transmission or reflectance responses areanalyzed by chemometric statistical analysis, an artificial neuralnetwork or a pattern recognition algorithm to correlate said responsesto at least one analyte, at least one bacterial species, or acombination thereof.
 12. A method for analyzing a food, blood, semen,sputum, mucous, feces, soil or water sample for a bacterial contaminantcomprising depositing a food, blood, semen, sputum, mucous, feces, soilor water sample onto or into the medium of the apparatus of claim 1 andmonitoring spectroscopic, transmission or reflectance responses of theplurality of chemoresponsive dyes thereby analyzing a food, blood,semen, sputum, mucous, feces, soil or water sample for a bacterialcontaminant.